Project Summary ? Project 3 Epigenetic regulators play key roles in controlling transcriptional programs in normal homeostasis and in fate specification. These regulators are frequently altered in cancer and thus offer potential targets for chemotherapeutic treatment. Project 3 is investigating two epigenetic regulators, BMI1 and PRMT5, whose upregulation in lung adenocarcinomas (LUAD) and higher-grade glioma, including glioblastoma multiforme (GBM), correlates with poor prognosis. Aim 1 focuses on BMI1, which acts as member of a polycomb repressor complex 1 (PRC1) to maintain the self-renewal capacity of adult stem cells and to regulate lineage choice in stem and progenitor cells. These observations suggest that BMI1 inhibition might effectively target cancer stem cells (CSCs). Using mouse models, Project 1 has shown that BMI1 inactivation at the time of tumor initiation greatly suppresses the progression of Kras:tp53 mutant LUAD, resulting in a significant extension of lifespan. Notably, K-ras mutations occur in approximately 30% of human LUAD and targeted therapies do not exist for these tumors. Thus, Bmi1 inhibition could offer a new strategy to target this disease. Experiments in Aim1 will determine the mechanism(s) by acute Bmi1 deletion impedes LUAD progression; in particular, they will test the hypothesis that this is caused by derepression of master differentiation regulators. Additionally, single tumor cell mRNA sequencing, in collaboration with Aviv Regev's laboratory, will determine how BMI1-deficiency alters the subpopulations of cells within Kras;tp53 LUAD especially the CSCs, complementing studies in Program 1. Final Aim 1 studies will model treatment of human disease by establishing whether acute Bmi1 deletion in existing autochthonous LUAD can achieve tumor suppression. Aim 2 investigates PRMT5, which accounts for the majority of symmetric dimethylation arginine (SDMA) modifications in vivo. In preliminary studies, Project 3 identified PRMT5 as the top hit in parallel in vivo and in vitro screens for glioma epigenetic regulators. Further analyses showed that suppression of PRMT5 by knockdown, or treatment with a PRMT5 inhibitor (PRMT5i) EPZ015666, caused cell cycle arrest and cellular senescence in GBM cells. This suppression correlates with widespread changes in expression levels, and also splicing alterations, which almost exclusively reflect elevated levels of detained introns (DIs). Aim 2 studies have two goals. First, they will identify the transcript changes associated with PRMT5i sensitivity and the core regulators that mediate these events, using a combination of gene expression analyses and a genome-wide dCas9-gene activation screen and complementing studies in Program 2. Second, they will directly address the therapeutic capacity of PRMT5i. A collaborative effort with Jun Qi's laboratory will generate PRMT5i with improved potency. Finally, since preliminary studies show that EPZ015666 effectively targets Kras;tp53 mutant LUAD cell lines, pre-clinical trials will assess the efficacy of PRMT5i in treating autochthonous LUAD tumors.